Role of immune cells in skin reinnervation by collateral sprouting after peripheral nerve injury

Peripheral nerve injury can result in loss of cutaneous sensory function, with associated defects in protective reflexes and tactile function, as well as neuropathic pain. While regeneration from injured nerve is the best- studied mechanism of reinnervation following injury, it is inherently slow even with surgical augmentation. Collateral sprouting from neighboring uninjured neurons represents an additional mechanism for recovery. Although axonal regeneration has been studied extensively, much less is known about the mechanistic basis or functional consequences of collateral sprouting. After peripheral nerve injury, macrophages are recruited into or proliferate within the injured nerve stump and areas undergoing Wallerian degeneration, where they play pivotal roles in phagocytic removal of neuronal and myelin debris and facilitate the regeneration of injured axons. However, changes and potential functions of macrophages in denervated skin after nerve injury remain less studied. In this project, we will explore the hypothesis that macrophages promote skin reinnervation by collateral sprouting of uninjured nociceptors after peripheral nerve injury. In Aim 1, we will characterize changes in the abundance, localization, morphology, and phenotype of macrophages in denervated skin over time and define the anatomical relationship of these cells to sprouting nociceptive fibers. In Aim 2, we will test the hypothesis that macrophages are essential for normal nociceptor sprouting and functional sensory recovery by quantifying sprouting-mediated skin reinnervation and behavioral sensory recovery after SNI surgery in mice in which macrophages have been globally or locally depleted. In Aim 3, to define the mechanisms by which macrophages promote collateral sprouting after nerve injury, we will assess whether macrophage depletion alters peripheral nerve injury-induced upregulation of sprouting-associated genes (SAGs) in spared nociceptive neurons. We also will use in vitro DRG neuron culture to examine the impact of prior in vivo injury, with or without macrophage depletion, on neurite outgrowth and branching. Finally, we will ask whether in vitro exposure of DRG neurons to either paw skin or macrophages isolated after nerve injury alters axon outgrowth or branching. Success in this proposal will allow us to elucidate the importance of cutaneous macrophages in anatomical and functional sensory recovery by collateral sprouting. It will also help to identify candidate transcriptional and cell biological changes driving these effects and establish a system with which to subsequently identify the responsible secreted macrophages effectors. Based on our findings, targeting of the recruitment, differentiation, survival, or effector function of macrophages might be explored as therapeutic strategies to promote the progression of sensory recovery and/or limit unwanted events such as chronic pain after peripheral nerve injury.